Exosomal circSTRBP from cancer cells facilitates gastric cancer progression via regulating miR‐1294/miR‐593‐3p/E2F2 axis

Abstract CircRNAs represent a new class of non‐coding RNAs which show aberrant expression in diverse cancers, such as gastric cancer (GC). circSTRBP, for instance, is suggested to be overexpressed in GC cells and tissues. However, the biological role of circSTRBP in the progression of GC and the potential mechanisms have not been investigated. circSTRBP levels within GC cells and tissues were measured by RT‐qPCR. The stability of circSTRBP was assessed by actinomycin D and Ribonuclease R treatment. Cell proliferation, migration, invasion and in vitro angiogenic abilities after circSTRBP knockdown were analysed through CCK‐8 assay, transwell culture system and the tube formation assay. The interaction of circSTRBP with the predicted target microRNA (miRNA) was examined by RNA immunoprecipitation and luciferase reporter assays. Xenograft tumour model was established to evaluate the role of exosomal circSTRBP in the tumour formation of GC cells. circSTRBP was upregulated in GC cells and tissues, and there was an increased level of circSTRBP in GC‐derived exosomes. circSTRBP in the exosomes enhanced GC cell growth and migration in vitro, which modulates E2F Transcription Factor 2 (E2F2) expression through targeting miR‐1294 and miR‐593‐3p. Additionally, exosomal circSTRBP promoted the tumour growth of GC cells in the xenograft model. Exosomal circSTRBP is implicated in the progression of GC by modulating the activity of miR‐1294/miR‐593‐3p/E2F2 axis.

treat, and systemic chemotherapy is generally applied to prolong the life span of the patients. 1,3vancement in molecular technology has enabled the identification of biomarkers for the diagnostic and prognostic purposes, as well as the biomarkers with the potential to inform treatment response and disease recurrence.Circular RNAs (circRNAs), for instance, have been extensively studied as potential cancer markers in recent years. 4,5CircRNAs represent a novel class of functional non-coding RNAs (ncRNAs), and over 100,000 unique circRNAs have been identified in the human transcriptome. 6,7ese ncRNAs are single-stranded and covalently closed molecules generated from precursor mRNAs through the back-splicing mechanism. 8Compared with their linear counterparts, circRNAs are highly stable with an extended half-life.Different groups of circRNAs are often expressed within specific cells or tissues. 8In addition, circRNAs can be exported outside the cell through exosomes and are secreted by most cells into biofluids such as blood, urine and saliva.
Numerous studies have shown the biological significance of circRNAs during tumour genesis and development. 4,5For example, circ_0009172 was reported to suppress GC progression by regulating miRNA-485-3p/NTRK3 axis. 9Circ-ITCH acts as a tumour suppressor to inhibit the metastasis of GC by targeting miR-199a-5p/Klotho axis. 10In addition, circRAB31 functions as a molecular sponge for miR-885-5p to suppress the malignancy of GC cells via modulating PTEN/PI3K/AKT pathway. 11Nevertheless, the functional roles of most circRNAs in the malignant progression of GC remain unclear.Genome-wide high-throughput sequencing of circRNAs has revealed the profile of circRNA deregulation in GC patients. 12circSTRBP (also known as hsa_circRNA_104907 or hsa_circ_0001888), was found to be upregulated within GC cells and tissues. 13However, the functional engagement of circSTRBP in dictating the malignancy of GC cells and in the progression of GC has not yet been clarified.In our study, we explored the functional role of circSTRBP in regulating the malignant progression of GC using in vitro and in vivo experiments.We revealed that circ-STRBP interacted with hsa-miR-1294 (a miRNA that was reported to be underexpressed in GC [14][15][16] ) and hsa-miR-593-3p.Both miR-NAs could negatively regulate E2F Transcription Factor 2 (E2F2) expression, an oncogene involved in the development of GC. 17 We further showed that circSTRBP was secreted in the exosomes by GC cells to promote cancer progression.

| Human GC tissues
A total of 80 pairs of GC tumour specimens and matched normal tissues were obtained by surgery from GC patients at Bozhou Hospital affiliated with the Anhui Medical University.Each sample was evaluated by two independent pathologists.This study gained approval from Bozhou Hospital affiliated with the Anhui Medical University Ethics Committee.All cases provided informed consent.was utilized in cell transfection experiment in line with specific instructions.Forty-eight hours after the transfection, the cells were harvested for further functional assays.

| Exosome isolation
Total Exosome Isolation Reagent (from serum) (4478360, Invitrogen, Shanghai, China) was adopted for isolating exosomes from cultured cells. 1 × 10 7 HGC27 cells or GSE-1 cells were cultured for 72 h, and the supernatant was collected and mixed with Total Exosome Isolation Reagent in 10:1 ratio (volume).The sample was mixed and subjected to 30-min incubation under 4°C, followed by a 10-min centrifugation at 10,000 g under ambient temperature.The exosomal pellet was reconstituted into 1 × PBS (400 μL) and preserved under −20°C prior to use.

| Immunofluorescence staining
After the extraction of cancer cell exosomes, the samples were conjugated by Dil (red) dye (72485, Sigma, Shanghai, China) before the addition into AGS and HGC-27 cells overnight.Incubation.The location of Dil-conjugated exosomes was observed under the Leica AM6000 microscope (Leica, Wetzlar, Germany).

| Cell Counting Kit-8 assay
CCK-8 assay kit (HY-K0301; MedChemExpress, Shanghai, China) was used for measuring cell growth in line with specific protocols.
After transfection, cells were inoculated into the 96-well plates for indicted culture periods.CCK-8 reagent (20 μL) was added into every well and the plate was incubated for another 2-h period under 37°C.
The measurement of absorbance (OD) value was conducted using a microplate reader at 450 nm.

| Fluorescence in situ hybridization (FISH)
HGC-27 and AGS cells were fixed and permeabilized before hybridization.The permeabilized cells were hybridized with Cy-3-labelled circSTRBP probes (RiboBio, Guangzhou, China) for a 16-h period under 37°C.The cells were then rinsed in saline-sodium citrate buffer under 42°C for three times, followed by 30-min incubation with Tween® supplemented within PBS that contained 5% BSA under ambient temperature.The Fluorescent In Situ Hybridization Kit (R1067, RiboBio, Guangzhou, China) was employed to detect the hybridization signals.

| RNA pull-down assay
Lipofectamine RNAiMAX Transfection Reagent (13778150, Invitrogen, Shanghai, China) was used to transfect NC probe (50 nM) or biotinylated miRNA mimic into AGS and HGC27 cells 48 h posttransfection; the cells were harvested and lysed, followed by the incubation with C-1 magnetic beads (65001, Life Technologies, Shanghai, China) for a 2-h period under 4°C.TRIzol (15596026, Invitrogen, Shanghai, China) was used to extract the RNA sample for subsequent qRT-PCR quantification.

| Luciferase reporter analysis
The luciferase reporters containing wild-type or mutated binding sites were prepared by the GenePhama (Shanghai, China).The reporters were delivered into HGC-27 and AGS cells by Lipofectamine 2000 reagent (11668019, Invitrogen, Shanghai, China) together with miR-NC or miRNA mimic.After 48 h, the Luciferase Reporter Gene Detection Kit (LUC1-1KT, Sigma, Shanghai, China) was used for the determination of luciferase activities in line with specific protocols.

| RNA immunoprecipitation
AGS and HGC27 cells were lysed with the cell lysis buffer in Magna RIP® RNA-Binding Protein Immunoprecipitation Kit (17-700, Millipore, Temecula, CA, USA) following specific instructions.
The lysates were then incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (ab156870, Abcam) or nonspecific IgG antibody (ab37450, Abcam) for 18 h under 4°C.TRIzol (15596026, Invitrogen, Shanghai, China) was used to extract the immunoprecipitated RNAs, followed by qRT-PCR detection.

| Calcein acetoxymethyl ester/propidium iodide (calcein-AM/PI) staining
To examine cell viability and cell death, a cell viability/death double staining kit (04511, Sigma, Shanghai, China) was used for simultaneous labelling of viable and dead cells.In brief, cells were washed with PBS and adjusted to the cell density at 1 × 10 5 cells/mL.One hundred microlitres of assay solution was mixed with 1 mL of cell suspension for the staining at 37°C for 15 min.After the staining, the cells were viewed under a fluorescence microscope with 490 nm excitation laser to simultaneously monitor the viable and dead cells.Living cells produce green fluorescence due to the conversion of calcein acetoxymethyl ester (calcein-AM) to calcein, while dead cells produce red fluorescence as a result of DNA staining by propidium iodide (PI).

| circSTRBP shows an elevated expression level in GC cells and tissues
We initially retrieved the circRNA profiling data of GC and normal samples from the public database (GSE83521 and GSE93541 microarray data).Gene expression analysis revealed the differentially expressed circRNAs in GC tissues compared with the normal specimens (Figure 1A).Among these circRNAs, more than 200 were upregulated (labelled as GSE83521-UP and GSE93541-UP, respectively) with the fold change >2 (p < 0.01).Hsa_circ_0001013 (hsa_circRNA_000684) and circSTRBP (hsa_circRNA_104,907/hsa_ circ_0001888) are the two commonly upregulated circRNAs in the two datasets (Figure 1B).We also collected 80 clinical samples of GC tumours and adjacent normal tissues to examine hsa_circ_0001013 and circSTRBP expression.Both circ_0001013 and circSTRBP showed significant upregulation in GC tumour specimens (both p < 0.001; Figure 1C,D).Since the functional role of circ_0001013 has been characterized in GC by a recent study, 18 we focused on the functional engagement of circSTRBP in GC progression in our study.To determine the prognostic significance of circSTRBP in GC, we examined the clinicalpathological features of 80 GC cases and the associations with circSTRBP expression level (Table 1).1H).After the blocking of transcription by actinomycin D, STRBP mRNA level showed a significant decrease within 24 h, while no significant change was observed for circSTRBP expression (Figure 1I).The above results demonstrated that circ-STRBP is substantially elevated within GC cells and tissues, which is related to the poor survival in GC patients.

| circSTRBP is packaged into exosomes from GC cells
We next examined whether circSTRBP is secreted into the exosomes by GC cells (HGC27).Figure 2A shows the transmission electron microscopy (TEM) image of exosomal sample isolated from the supernatant of GC cell culture.Figure 2B displayed the size distribution of GC cell-derived exosomes, which was determined by the nanoparticle tracking analysis (NTA).The WB assay for CD63, ALIX and TSG101 protein levels in exosomes derived from normal cells (GSE-1) and GC cells (HGC27) showed that those exosomal markers were expressed in both exosomes (Figure 2C).RT-qPCR quantification suggested that there was more abundant circSTRBP within the exosomes derived from GC cells compared with the normal cellderived exosomes (Figure 2D).We next incubated GC cells with Dillabelled exosomes isolated from GC cell culture, and the Dil-labelled exosomes could be observed inside the two cell lines after incubation, as shown by phase-contrast microscopy images (Figure 2E).
Collectively, these data suggest that circSTRBP is secreted into GC cell-derived exosomes.

| Exosomal circSTRBP promotes GC cell growth and mobility
We next investigated the role of exosomal circSTRBP in GC cells by knockdown experiments.circSTRBP level was repressed after si-circSTRBP (siRNA targeting circSTRBP) transfection in HGC27 cells when compared to the cells with si-NC transfection (control siRNA) (p < 0.01; Figure 3A).Similarly, in the exosomal samples derived from GC cells with si-circSTRBP transfection, circSTRBP expression level was significantly reduced (p < 0.001; Figure 3B).Moreover, we incubated AGS and HGC-27 cells with exosomal samples isolated from si-circSTRBP-treated (exo-si-circSTRBP) or si-NC-treated (exo-si-NC) HGC-27 cells.In both cells, circSTRBP expression level was significantly reduced after the incubation with exo-si-circSTRBP sample compared with exo-si-NC sample (p < 0.001; Figure 3C).As revealed by CCK-8 assay, exo-si-circSTRBP group showed a decreased cell viability compared with exo-si-NC group (p < 0.01; Figure 3D).The analysis of cell viability and death by calcein-AM/PI staining showed that the number of calcein-AM-positive cells (live) decreased and PI+positive (dead) cells increased in the exo-si-circSTRBP group (p < 0.01; Figure 3E).Transwell assays showed that incubation with exo-si-circSTRBP impaired the cell invasion and migration in both AGS and HGC-27 cells (p < 0.01; Figure 3F,G).Furthermore, we evaluated the angiogenic potential and found that exo-si-circSTRBP group displayed an impaired tube formation capacity compared with exo-si-NC group (p < 0.01; Figure 3H).WB analysis also revealed that the incubation with exo-si-circSTRBP increased E-cadherin level (epithelial marker), whereas the expression levels of angiogenic markers (VEGFA and N-cadherin) were repressed (Figure 3I).

| miR-1294 and miR-593-3p could target E2F2
Targetscan database prediction of the potential targets of miR-1294/miR-593-3p and the analyses with the genes upregulated in GC using the public datasets identified E2F2 as the common target of the two miRNAs (Figure 6A). Figure 6B,C show the binding sites predicted between E2F2 mRNA and miR-593-3p/miR-1294, respectively.AGS and HGC-27 cells were transfected with luciferase reporter vectors that contain the WT or MUT sequence of E2F2 mRNA.miR-1294 and miR-593-3p mimics markedly reduced luciferase activities of WT E2F2 reporter, whereas no inhibition was seen in cells transfected with the MUT reporter (p < 0.01, p < 0.001, separately; Figure 6D).Additionally, both miR-593-3p miR-1294 overexpression decreased E2F2 protein levels within these two cell lines (p < 0.01; Figure 6E).The exo-si-circSTRBP was able to suppress E2F2 protein level, which was antagonized by miR-593-3p and miR-1294 inhibitor (Figure 6F).In the 80 pairs of GC tumour and normal specimens, E2F2 mRNA showed an upregulation in the cancerous tissues (p < 0.001; Figure 6G).Pearson correlation analyses further showed that E2F2 level was negatively related to the expression of miR-593-3p or miR-1294 (both p < 0.001; Figure 6H,I).In contrast, E2F2 level displayed a positive association with circSTRBP level (p < 0.001; Figure 6J).

| Exosomal circSTRBP promotes the tumorigenesis of GC cells in vivo
To further demonstrate the pro-tumorigenic effect of exosomal circ-STRBP in vivo, nude mice were injected with AGS cells, and the mice were assigned into exo-si-circSTRBP group (intratumorally injected with exosomes extracted from circSTRBP siRNA-transfected AGS cells) and exo-si-NC group (intratumorally injected with exosomes from AGS cells transfected with the control siRNA).The administration of exo-si-circSTRBP significantly attenuated the tumour formation of AGS cells in the nude mice (p < 0.05; Figure 8A,B).Besides, IHC staining of Ki-67 and E2F2 in the xenograft tumours revealed that the reduction in E2F2 level in the exo-si-circSTRBP group was accompanied by the lowered level of Ki-67 staining (p < 0.05; Figure 8C).
We further examined the relative RNA levels of circSTRBP, miR-1294, miR-593-3p and E2F2 in tumour tissues.Compared with the exosi-NC samples, tumour tissues from the exo-si-circSTRBP group expressed lower levels of circSTRBP and E2F2 and the increased levels of miR-1294 and miR-593-3p (p < 0.05; Figure 8D).These data further corroborate the oncogenic role of exosomal circSTRBP in promoting the tumorigenesis of GC cells in vivo.

| DISCUSS ION
GC is a common malignancy and a leading cause of cancer-associated mortality globally. 1Understanding the molecular basis of GC progression is the key for early diagnosis and the formulation of novel treatment schemes.Emerging evidence pinpoint that the deregulation of circRNAs contributes to cancer development and progression.circRNAs have been characterized as a novel group of ncRNAs showing aberrant expression within several cancers such as GC. 19e work explored the functional engagement of exosomal circ- Previous evidence indicates that circRNAs are enriched in cancer cell-derived exosomes. 20,21These extracellular vesicles play pivotal roles in cellular communication by carrying a variety of cargo, including DNA, RNA, proteins and lipids, which could be taken up by the neighbouring cells to impact the functional properties. 20In cancers, exosomes can function to establish a favourable microenvironment for disease progression and metastases. 22,23The study by Li et al. 24 provided solid evidence supporting the enrichment of circRNAs within exosomes derived from cancer cells.In addition, exosomal circRNAs in the serum sample have been proposed to serve as biomarkers to discriminate colorectal cancer (CRC) cases from normal subjects, and there was an association between the expression levels of exosomal circRNAs and tumour mass. 24Following that, numerous studies have examined the association between exosomal circRNAs in serum and the clinical progressions in patients suffering hepatocellular cancer (HCC), GC and pancreatic cancer.6][27][28][29][30][31] Our results added novel evidence regarding the overexpression of circSTRBP The most well-established function of circRNAs is to serve as molecular sponge to limit the activity of miRNA targets.CDR1as, the first circRNA with characterized biological activity within exosomes, was found to act as a sponge of exosomal miR-7. 24This study suggests that circRNAs in the exosomes preserve the activity to sponge miRNA and regulate the expression of downstream mRNA targets in the recipient cells.Notably, a single circRNA may contain multiple seeding sequences for adsorbing distinct miRNAs, which may function as a tumour-suppressive or an oncogenic factor in different scenarios. 32cording to our results, exosomal circSTRBP plays an oncogenic role via inhibiting miR-1294 and miR-593-3p in GC cells.5][16] In agreement with these studies, we showed that miR-593-3p and miR-1294 suppressed GC viability and aggressiveness.Furthermore, we showed that the two miRNAs target E2F2, a transcription regulator whose dysregulation can lead to aberrant cell proliferation and cell cycle progression. 33F2 was previously reported to be overexpressed in GC and miR-31 was found as a regulator of E2F2 within GC cells.E2F2 silencing impaired the cell growth, invasion and migration of GC cells.34 Thus, our data are consistent with the reported pro-tumorigenic role of E2F2 and provide novel insights into the regulation of E2F2 expression by circSTRBP/miR-593-3p/miR-1294 axis in GC.

AGS cells ( 1 ×
10 7  ) were suspended in 200 μL normal saline and subcutaneously injected into right flanks of BABL/c male nude mice (6-8-week-old; Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).The mice were then randomly assigned to exosi-circSTRBP or exo-si-NC group (n = 6/group).On Day 7 following tumour cell injection, each mouse was intratumorally injected with 10 μg of exosomes extracted from circSTRBP siRNA-transfected AGS cells or AGS cells transfected with the control siRNA.All the mice were sacrificed by cervical dislocation on Day 28 and the tumour tissues were harvested for weight determination and subsequent analyses.

1
circSTRBP is highly expressed within GC cells and tissues.(A) Volcano plot showing the downregulated (in green) and upregulated (in red) circular RNAs in GC samples in comparison with normal tissues.The analyses are from GSE83521 and GSE93541 datasets.(B) The upregulated circRNA sets of GSE83521 and GSE93541 shared two common elements: hsa_circ_0001013 and circSTRBP.(C) circ_0001013 expression in 80 pairs of GC and para-cancerous samples.(D) circSTRBP expression in 80 pairs of GC and para-cancerous samples.(E) Overall survival of GC patients in circSTRBP high and low expression groups.(F) Relative expression of circSTRBP within GC cell lines (HGC27, AGS, MKN-45 and MKN74) and normal gastric epithelial cells (GSE-1).(G) Schematics of circSTRBP sequences and structures.(H) Relative RNA levels within HGC-27 and AGS cells with/without RNase R exposure.(I) Relative RNA levels with or without actinomycin D treatment in AGS and HGC27 cells.*p < 0.05; ***p < 0.001.WANG et al.

F I G U R E 2
Package of circSTRBP in GC cell-derived exosomes.(A) The transmission electron microscopy (TEM) image of GC cell (HGC27)-derived exosomes.Exosomes are marked by red arrows.(B) The size distribution of exosomes in the supernatant of GC cell culture.(C) Relative expression levels of CD63, TSG101 and ALIX in exosomal samples derived from normal gastric epithelial cells (GSE-1) and GC cells (HGC27).(D) circSTRBP levels within the exosomes derived from normal gastric epithelial cells (GSE-1) and GC cells (HGC27).(E) AGS and HGC-27 cells were incubated with Dil-labelled exosomes and the uptake of the exosomes was imaged under the fluorescence microscope.*p < 0.05; **p < 0.01; ***p < 0.001.

F I G U R E 7
Exosomal circSTRBP promotes GC progression via modulating E2F2.(A) E2F2 levels within HGC-27 and AGS cells after the transfection of empty vector or E2F2 expression vector.(B-F) HGC-27 and AGS were divided into exo-si-NC, exo-si-circSTRBP or exo-si-circSTRBP+E2F2 transfection groups.(B).CCK-8 viability assay, (C) Transwell migration assay, (D) Transwell invasion assay, (E) Tube formation assay and (F) WB analysis of E-cadherin, N-cadherin, β-Actin and VEGFA levels in each experimental condition.**p < 0.01; ***p < 0.001 versus exo-si-NC.^p < 0.05; ^^p < 0.01; ^^^p < 0.001 versus exo-si-circSTRBP.inGC tumours and characterized circSTRBP as a tumour-promoting factor secreted in the exosomes by GC cells.Our data also indicated that circSTRBP may be employed as a prognostic biomarker for GC patients as its high expression level was associated with a poorer overall survival.Currently, there is no reports regarding the functional role of circSTRBP in other types of malignancies.Whether the exosomal circSTRBP could serve as a biomarker for GC diagnosis and prognosis needs to be validated in a large cohort of clinical samples.Furthermore, whether exosomal circSTRBP can shape the tumour microenvironment in GC warrants future investigation.

F I G U R E 8
is enriched in exosomes derived from GC cells.Exosomal circSTRBP may promote the viability and the aggressiveness of GC cells via miR-1294/miR-593-3p/E2F2 axis.Nevertheless, the clinical potential of circSTRBP as a circulatory biomarker for GC diagnosis requires further clarification.More investigations are needed to unravel the mechanisms underlying the packaging and trafficking of exosomal circSTRBP, as well as the biological functions in the tumour microenvironment.Further, future efforts are required to optimize the delivery method of circSTRBP targeting siRNA in animal models in order to evaluate the effect of circSTRBP silencing on GC progression.AUTH O R CO NTR I B UTI O N S Yin Wang: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); validation (equal); visualization (equal); writing -original draft (equal).Deke Li: Methodology (equal); writing -review and editing (equal).Rong Zou: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); validation (equal); visualization (equal); writing -original draft (equal).Xiankui Gao: Data curation (equal); formal Exosomal circSTRBP promotes GC cell growth in vivo.(A and B) The volume (mm 3 ) and weight (mg) of the xenograft tumours in exo-si-NC and exosi-circSTRBP groups.(C) IHC staining of Ki-67 and E2F2 in the xenograft tumour sections.(D) Relative RNA expression of circSTRBP, miR-1294, miR-593-3p and E2F2 in tumour samples of exo-si-NC or exo-si-circSTRBP groups.*p < 0.05; **p < 0.01; ***p < 0.001.

2.10 | Tube formation assay
The circ-STRBP expression values in cancerous tissues were considered as binary variable (high or low), with the median value as threshold to classify the high-and low-level groups (n = 40 cases in each category).The elevated circSTRBP level was related to more distant me- highest expression (p < 0.05 or p < 0.001; Figure1F).As illustrated in Figure1G, circSTRBP is produced by the back-splicing of exons 2, 3, 4 and 5 from pre-STRBP mRNA.To confirm the stability of circSTRBP, we carried out Ribonuclease R (RNase R) digestion as well as actinomycin D treatment.RNase R exposure significantly reduced STRBP mRNA level, whereas circSTRBP level remained unchanged (p < 0.001; Figure